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avdt'btofMfiilm-Si J& isNVIKONMKN'USI. BESEAKCH 2, 1-10 (1968) ;er of Industrial'Hygiene j >ccupational Diseases ; Czechoslovakia j 'r coniosis Research f | frican Council for ific and Industrial oh j, ! I Renal Excretory Mechanisms of Heavy Metals I. Transtubular Transport of Heavy Metal Ions in the Avian Kidney J. VOSTAL AND J. HELLER Institute of Industrial Hygiene and/ Occupational Diseases, Praha, Czechoslovakia Received June 6, 1967 sbwg, Soidk Africa f Inulin, as & reference glomerular substance, was injected simultaneously with radio active isotopes of copper, cobalt, chromium, manganese, molybdenum, mercury or lead .y 'Jnion Congress England ions, as well as with cadmium in the form of cadmium cysteinate hydrochloride, into the renal portal circulation of the chicken. Radioactivity in all studied metals, inde pendent of valence or positive or negative charge of the ions, was demonstrated in ielson fractionated urine collections before the first detectable traces of inulin. All studied rk University Medical heavy metals are therefore considered to be similar to sodium and other essential ions rk. New York 10018 in being able to pass into the urine not only by glomerular filtration but also by direct transport across the tubular wall. The possible significance of this transtubular trans of Occupational , Finland ;netla !a Degli Studi Italy lford, Jr. \y of Cincinnati i of Medicine ti, Ohio 46m ibcrtsou Department of Uure Canada R. Boucofc Sturgis : Medical College nsylvanta ihia, Pennsylvania port in the renal excretory mechanism of heavy metals, in relation to tlieir plasma diffusible fractions, is briefly discussed. t The pharmacology of heavy metals, in view of their extensive use in industry and their increasing presence in the external .environment, has attracted con siderable attention. Results of recent studies have been summarized (Passow et al., 1961). The general aspects of the mechanisms of renal excretion have not, however, been studied to any great extent so far, and in some respects even basic 1 data are lacking. Knowledge of excretory mechanisms may be expected to con tribute^understanding of basic principles of evaluation of individual or population exposure and may even afford the possibility of therapeutic alteration of such regulatory mechanisms. Although individual heavy metals differ considerably in their chemical re- < activity, they are practically all able to bind with a number of organic molecules. I The high degree of their interaction with ligands present in erythrocytes and/or i plasma proteins, however, considerably limits the use of classical clearance of Occupational Hungary lk Idskovic Institute Yugoslavia nor ichool of Hygiene rapical Medicine England tiiib.'trn o/ Mines Research tones burg. South Africa ruchi Institute of ini Health i, Japan methods (Smith, 1955) in the study of their renal excretion. If ultrafiltration I through the glomerular membrane and/or permeation through the tubular wall in ! both directions be considered (the two most important general processes in the transport of any substance between blood and urine) then it may be agreed that the participation of glomerular filtration in the mechanism of renal excretion has been demonstrated for a number of metals (Wills, 1953 for uranium; Hursh et al., 1960, for radium; Collins et al., 1961, for chromium; Vostal, 1963, for lead, etc.) and predicted in others (Simmonds, 1942; Rodin and Crowson, 1962; or Gayer et al., 1962, for mercury, etc.). On the other hand, the predominance of tubular secretory mechanisms in metal excretion by the kidney has been stressed for mer cury (Bergstrand el al., 1959; Passow et al., 1961; Berlin and Gibson, 1963; Mam- 196S by Academic Press Inc. 1 I f j I ! ? \\ N36904 2 v o s t Al a n d h e l l e r bourg and Raynaud, 1965, etc.) and the experiments carried out by stopflow tcchnique in clogs by Gayer et al. (1962) tended to demonstrate tubular reabsorp tion of filtered mercury in the kidney. Similar behavior of lead ions as well was observed in acute exposure experiments on dogs (Vostal, 1963). The question, therefore, whether heavy metal ions are able to be transported across the cellular structures of the renal tubule seemed to be important for demonstrating tubular component participation in the general mechanisms of heavy metal excretion by the kidney. Renal portal circulation in avian kidney has been used in this investigation to study the possibilities of transtubular transport of heavy metals. EXPERIMENTAL APPROACH The transport of sodium and other ions across the tubular wall not only from the lumen into peritubular fluid but also in opposite direction was demonstrated by Chinard and Enns (1955), after simultaneous injection of radioisotopes to gether with a glomerular substance directly into renal artery of the dog. A glomer ular substance which does not penetrate the tubular wall can appear in urine only after ultrafiltration through the glomerular membrane. Earlier appearance of the simultaneously injected isotope must mean that this ion was able to pass ART. RENAL !. P.'i' < :-:*i r < ! - Ulbui:.r !:. 11 ' 21 lit! ' in- liru'-iu!ilire body * ,-lu.i.d if.. Fio. 1. Schematic drawing showing Chinard's technique of transtubular transport testing in the mammalian kidney. Tested substance (black arrows) is injected together with glomer ular ^substance (striped arrows) simultaneously into the renal artery of the dog kidney. through the tubular epithelium distal to the glomerulus i.e. by-passing, with the postglomerular blood, part or parts of the nephron more rapidly than the filtrate flowed through the tubular lumen and thus reached, via the peritubular capil laries, the outer wall of tubular ceils and collecting ducts earlier. A concentration gradient was thereby formed between the peritubular fluid and the tubular lumen, enabling transport of the injected radioisotope across the tubular wall (Fig. 1). Fie. 2. ?' *i 1 Mil .'irnnvO into Malt* chi 't*cd :is ox mg kg riic outer DUP050312353 it by stopflow iIar reabsorpns as well was The question, k s the cellular rating tubular il excretion by ivestigation. to not only from i demonstrated iioisotopes todog. A glomer)pear in urine ier appearance is able to pass TRANSTUBULAR TRANSPORT OP HEAVY METALS The circulatory differences in mammalian and avian kidney seemed to offer better conditions for testing the existence of transtubular transport. Renal blood supply in birds is mediated by both the renal artery and the vena portae renalis, which simultaneously drains the venous system of the pelvic leg on the same side (Sperber, 1948). Blood flow of the portal circulation omits the glomerulcs. Its branches enter close to the vasa efferentia of the glomerulcs, directly into the common peritubular capillary net. If a substance being studied is transported across the tubular wall, it appears in ureteral urine after injection into portal circulation much earlier than a substance excreted only by glomerular filtration, which would be brought to the glomerular membrane only after recirculation through the en tire body (Fig. 2). Moreover, the participation of other factors is completely ex cluded (Vostal and Heller, 1963). V j.i transport testing her with glomerlog kidney. issing, with the ban the filtrate itubular capill concentration tubular lumen, wall {Fig. 1). Fra. 2. Schematic drawing showing transtubular transport testing in the avian kidney. Tested substance (black arrows) is injected together with glomerular substance (striped arrows) into the renal portal system of chicken. MATERIALS AND METHODS Male chickens, Gallus domesticus, variety White Leghorn, weight 2-3 kg, were used as experimental animals. They were anesthetised by sodium pentobarbital (30 mg/kg i.p.) and ureters were cannulated from cloaca or after perineal incision. The outer diameter of the polyethylene catheters, always inserted at the same dis- DUP050312354 .^^as^asaaiajaafei^ V ,. 5 jl \ i i I ? f i :s * . -; 1 . \ i ; i >I 4 v o s t Au a n d h e l l e b tance from the cloaca and tied in place, was such as to fit snugly into the ureters. To provide adequate urine flows intravenous injection of hypertonic 5% sodium chloride or 10% urea solution was started at the beginning of each experiment and supplemented by continual infusion as required during the course of experiment. Urine flow of 0.6-1.0 ml/kg/ininute from each side was considered adequate. When the urine flow was adequate, the leg vein--a branch of vena ilica externa-- on the side of the urine collection, was cannulated. After three control drops of urine directly from the cannula opening into small glass test tubes (arranged in an order similar to that used in stopflow technique) had been obtained, isotonic radioisotope solution in 0.9% N4C1 containing inulin was rapidly injected and collection of individual urine drops continued. The volume of injected test solution was always 2 ml; the injection took 1--2 seconds for completion. The radioactivity of the injected solution was in all instances the same, i.e. 100 /*Ci; the amount of simultaneously injected inulin used as the reference glomerular substance was 100 mg. Experiments with individual isotopes were carried out in groups of 4-6 ani mals. The integrity of the portal circulation was tested in all animals after the experiment by intraportal injection of 0.2 mg/kg phenol red in saline and the functional slate of portal circulation visually evaluated by observation of a con siderable excess of red color excreted in the urine from the experimental side (Campbell, 1960). Exceptional animals whose portal circulation could not be clearly demonstrated to be functionally sound, were not included in the study. As the radioactivity on the control side was regularly delayed and appearing with the inulin, after the first experiments corrections for excretion from the control side were not carried out. Radioactive lead 212Pb++ (Thorium B), carrier-free, was produced by electrodeposition in a radioemanator containing radiothorium (RdTh228, halflife 1.9 years). Electrodeposits were dissolved in 2n nitric acid p.a., neutralized and suitably diluted by 0.9% sodium chloride solution. Copper "Cu** (spec, activity 2.5 Ci/gm Cu) as well as freshly produced cadmium 11BCdw' (spec, activity cea 0.1 Ci/gm Cd), both in the form of chloride salts, were supplied by Zentralinstitut fur Kemforschung, Dresden, DDR. However, preliminary experiments showed that the carrier content was toxic for experimental animals, despite high specific activity of freshly produced radioisotope. Therefore, cadmium had to be injected as a mixture with cystein hydrochloride in 1:20 molar ratio (Vander, 1962). Manganese S!MnH (carrier-free) and mercury 203Hg,+ (spec, activity 50-70 mCi/gna Hg), both also in the form of chloride salts, were products of the Institute of Nuclear Research, Prague. Cobalt 5TCo++ (carrier-free) in the form of cobaltous chloride and hexavalent molybdenum 98Mo" (spec, activity 5 Ci/ gm Mo) in the form of ammonium molybdenate as well as chromium B1Crc' in the form of sodium chromate were obtained from Radiochemical Centre in Ammersham, England. Radioactivity of the individual drops of urine was meas ured in a well-type scintillation counter without further adjustments of the sample and, when necessary, corrections for radioisotope decay were made. Results were expressed in counts per minute per individual drop. Measurement time was se lected after radioactivity sampling demonstrated significant differences from the background, within a 5% error. Urine drops, after proper dilution in the same T) test tubes, were Hu m method (Harrison, 1 Figure 3 demon*!, of inulin and radioa chicken and indivkl and isotope. Quite si radioisotopes of otlu .available. These c> radioactivity appear: 5- O o O Fio. 3. Excretion i staataneous injection < of a chicken at the mo two figures represent tl separately from left ur total volume of urine appearance of dctcclnb: which does not pas.graphic presentntioi ferent radioisotopes were obtained by a t percents were calcul and the first evident: In Figs. 4 and 5 isotopes of heavy i studied metal ions, the ion, appear in portal circulation i merely by gloment other ions (Yostal a J I i DUP050312355  JgjvS.%AiA,\JX.' lAib&s&i&its. o the ureters. : 5fo sodium oeriment and f experiment. ed adequate. ,ca externa-- trol drops of (arranged in ned, isotonic injected and test solution radioactivity le amount of ince was 100 3 of 4-6 anials after the line and the ion of a con imental side ould not be ,he study. As pearing with i the control : : j j $ ' $ j : j T.RANSTUBULAR TRANSPOItT OF HEAVY METALS 5 test tubes, were then analysed for inulin by a micromodiiication of diphenylamine method (Harrison, 1942) with alkali treatment. RESULTS Figure 3 demonstrates a typical result of one of experiments in which a mixture of inulin and radioactive lead 2J-2Pb++ was injected into the portal circulation of a chicken and individual drops of subsequently produced urine analysed for inulin and isotope. Quite similar results were obtained in other experiments with lead and radioisotopes of other heavy metals, wherever their gamma-emitting isotopes were available. These experiments demonstrated that after simultaneous injections radioactivity appears in urine much earlier than simultaneously injected inulin, roduced by , ITh228, half- | , neutralized "Cu+* (spec. ` ;pec. activity by Zentralexperiments despite high m had to be j tio (Vander, , iec. activity \ iducts of the * in the form itivity 5 Ci/ .; um 61Cr&' in : tl Centre in . & was meas- t if the sample Results were ; lime was se- ces from the , tin the same ' Fic. 3. Excretion pattern of radioactive lead isotope () and inulin (A) after an in stantaneous injection of 100 jaCS JTb'"' together with 100 mg of inulin into the left leg vein of a chicken at the moment indicated by an arrow. Different symbols in this and the following two figures represent, the concentration of tested substances in single drops of urine collected separately from left ureter. Individual drops are on the abscissa expressed as a percentage of total volume, of urine excreted by the kidney in the time interval between injection and first appearance of detectable traces of inulin. which does not pass through the tubular wall in either of the two directions. For graphic presentation and comparison of individual experiments as well as dif ferent radioisotopes additional calculations were made. Although individual drops were obtained by a modified stopflow technique (Malvin et al., 1958) urine volume percents were calculated from the volume of urine obtained between the injection and the first evidence of measurable inulin concentrations. In Figs. 4 and 5 typical excretion patterns obtained with representative radio isotopes of heavy metals are shown. Both figures clearly demonstrate that all studied metal ions, independent of their valence or positive or negative charge of the ion, appear in the urine, after simultaneous injection with inulin into the portal circulation of chicken kidney, much earlier than a substance excreted merely by glomerular filtration. As in previous experiments with sodium and other ions (Vostfi.1 and Heller, 1963), the data demonstrate that the studied heavy I il ( i i -i DUP050312356 6 v o s t Al a n d h e l l e r metals ions, as well as cadmium cysteinate hydrochloride, are transported across the tubular wall distal to the glomerulus, in the chicken kidney. Significant in crease in radioactivity appeared at 32-62% of the urinary volume excreted during the interval between the injection and first traces of inulin in the urine. Although individual heavy metals differed in their excretion pattern, the recorded differ ences in level and extent of radioactivity excretion remained within limits of one ,1 Fin. 5. Cun; donum and system of ettiefc part of the n Therefore, d by possible i Fig . 4. Cumulative graph of typical excretion patterns of radioactive lead, mercury, cad mium (as cysteinate hydrochloride) and manganese after instantaneous injections with inulin into the renal portal system of chicken kidney at the time indicated by an arrow. In view ol in the appe; order. These differences may be primarily attributed to variable urine flow relative differences i to different efficacies of scintillation equipment for energetically different radi the quest lor ation levels of various isotopes. Also, the possible effect of various degrees of have for Ui< binding on plasma and tissue components, as well as possible differences in dif heavy rneta fusion coefficients should be considered in interpretation of the results. Current trnvnseular knowledge of the chicken kidney docs not allow evaluation of the role of each a- other os: DUP050312357 I sported across Significant inxcreted during line. Although icorded differ:i limits of one. TRANSTTJBULAR TRANSPORT OP HEAVY METALS "iii, mercury, cadrtions with inulin ine flow relative different radi 'ioiis degrees of fimniees in diffv*ult.s. Current fisc role of each Tic. 5. ^Cumulative graph of typical excretion patterns of radioactive copper, cobalt, molyb denum ana chromium ions after instantaneous injections with inulin into the renal portal system of chicken kidney at the time indicated by an arrow. part of the nephron reached by intraportal injection in the individual experiments. Therefore, different excretion patterns cannot, for the time being, be explained by possible differences in excretory mechanisms for the several metals. DISCUSSION In view of the fact that Chinard (1955) has demonstrated that time differences in the appearance of injected metals and inulin in urine cannot be explained by differences in diffusibility of these substances in tubular fluid in nephron lumen, the question remains as to significance the proof of transtubular transport might have for the study of renal excretion of metals. It can be generally assumed that heavy metals should be, except for their increased ability to be bound onto in travascular and/or intracellular ligands, excreted by the same renal mechanism as other essential cations and anions. These mechanisms might, however, differ DUP050312358 Jr v 4 i l -t, 8 * VOSTAL AND HELLER considerably in individual cases, as shown by the well known differences in the excretory mechanism of chemically rather similar ions as e.g. sodium, lithium and potassium, or calcium, strontium and magnesium on the other hand (Smith, 1953). The participation of a tubular component, evident in the excretion of the abovementioned essential cations, has been demonstrated for heavy metal ions by our results. Along with increased binding to plasma proteins--if we consider the fraction bound to blood corpuscles as not available for excretion--and therefore a decreased plasma diffusible fraction of heavy metals in blood, probably the importance of glomerular filtration in the mechanism of excretion of at least some representatives of the heavy metals group diminishes, and the higher binding ability of intracellular ligands of tubular cells may take precedence over the intravascular ligands. Rapid increase in the concentration of the metals studied, in peritubular capillary blood immediately after intraportal injection, permitted our demonstration of transtubular transport for all studied Heavy metal ions, although the quantitative degree of this transport differed considerably for in dividual ions. If the majority of all previously mentioned factors, probably responsible for differences found in excretion patterns, be neglected, one common factor of decisive importance for the existence of any transport in the living organism remains, i.e. the level of the diffusible fraction in blood. This diffusible fraction, consisting theoretically of ionized and unionized bound component, could be predicted for heavy metals on the basis of similar behavior of other ions in plasma. Its level, however, would probably remain under the level of our detectable limits (Berlin and Gibson, 1963; Vostal, 1963). It would depend not only on the metal-binding ability of various biological ligands as, e.g. amino-', carboxy-, and thiol-groups, where, the sequence of metal affinity might differ considerably (Passow et al, 1961), but would also be determined by the fact that some of these ligands arc on nondiffusible macromolecular tissue or plasma proteins while others are on diffusible molecules, e.g. natural amino- and other organic acids. The level of the diffusible component would therefore probably be conditioned by many complex factors already in physiological homeostasis some of which might be affected by additional toxic effects of heavy metals, in which, for example, aminoacids might change considerably (Thoelen and Pleteher, 1953; .Clarkson and Kench, 1956). The diffusible fraction may also pass from the vessels into the extracellular fluid and consequently across the cellular membrane into the intracellular fluid; it could then be easily transported into the tubular cells. The presence of specific macromolecular nondiffusible ligands in renal tubular cells with known high af finity for a number of heavy metals, competes thereafter for entering metal ions within the diffusible fraction. The resulting decrease in metal concentration in the diffusible fraction encourages further transport of metal ions into the cell from the extracellular fluid, following the concentration gradient. Although the steady state between the concentrations of diffusible metal and metal bound to non diffusible macromolecular ligands inside the cell is highly shifted in favor of the bound component, the diffusible fraction, although low, may come into contact with the cell membrane separating the tubular cell from the nephron lumen and, following the concentration gradient, penetrate into the tubular fluid (Fig. 6). duly l.y i 1 -t >t pu< >: ; the mat.; lit- U*ly ii;i Fig . 6. h ` ) fnllov-vi h may I-1 -ivy met uv. ami other ' "!i. but : ''at exte well a, i'Iu ImiI `!!S~ Use ' ' t toil of 1 DUP050312359 SS& Aik. V TBANSTUBULAR TRANSPORT OF HEAVY METALS 9 lifferences in the. iuin, lithium and ' id (Smith, 1953). on of the aboveletal ions by our we consider the n--and therefore od, probably the ction of at least < he higher binding jedence over the e metals studied, cction, permitted eavy metal ions, siderably for in- , s f y responsible for factor of decisive nism remains, i.e. ' action, consisting ' be predicted for plasma. Its level, ble limits (Berlin ,he metal-binding and thiol-groups, i (Passow et al., \ these ligands are iile others are on ; . The level of the ay many complex . ;ht be affected by ' aminoacids might nd Kench, 1956). the extracellular ' ntracellular fluid; ; reeence of specific ; h known high af- ' atering metal ions ; neentration in the ' into the cell from though the steady al bound to noned in favor of the come into contact iphron lumen and, fluid (Fig. 6). I This is probably not the only mechanism underlying the observed transtubular transport of metal ions, since after intrarenal administration, considerable dif fusion of free metal ion into the extracellular fluid may occur, reflecting the fact that binding to blood components is not yet in equilibrium. Consequent formation of a high concentration gradient in diffusible fractions considerably favors trans port across the tubular celj. The existence of a diffusible component formed mainly by organic bound metal ions has also been suggested to explain metal reabsorption through the intestinal wall (Saltman, 1965) and could well be one of the many renal mechanisms by which the heavy metal ions are excreted from the body into the urine. INTRAVASCULAR , INTRACELLULAR INTERSTITIAL INTRATUBULAR BLOOD EXTRACELLULAR TUBULAR FLUID - CELL TUBULAR LUMEN e BOUND O DIFFUSIBLE % LIGANDS Fio. 6. Schematic drawing showing possible transport of heavy metals diffusible fraction (O) following the concentration gradient across the renal compartments into the tubular ' / ; fluid. It may be concluded therefore that we have demonstrated that all studied heavy metal ions, i.e. copper, cobalt, chromium, molybdenum, manganese, mer- | eury, and lead as well as cadmium cysteinate hydrochloride are similar to sodium and other ions in being able to pass into the urine not only by glomerular filtration, but also by direct transport across the tubular wall. Whether or not and to . what extent this mechanism participates in the normal excretion of these metals, as well as the possibility of participation of active secretory processes in this . transtubular transport, still remain open questions. The approach here utilized , - mns useful for obtaining further information on the mechanisms of renal exrretion of metals. ;1 DUP050312360 10 VOSTAIx AND HELLER REFERENCES Ber c s t r an d , A., Fr iber g , L., Me n d el , L. a n d Ou ebl ad , E. (1950). The localization of sub cutaneously administered radio-active mercury in the rat Iddney. /. Ullrastructurc Research, 3, 234-240. Be r l in , M. an d Gid s o n , S. (1963). Renal uptake, retention and excretion of mercury. I. A study ill the rabbit during infusion of mercuric chloride. Arch. Environmental Health 6, 617-62li. Campbel l , D. E. S. (1960). Modification by bromcresolgreen or probenecid of the excretion and diuretic effect of three mercurial diuretics: Diurgin, Chlonnerodrin and Mercumntiliii. Acta pkarmacol. et toxicol. 17, 213-232. Ch in ar d , F. F. (1955). Comparative renal excretions of glomerular substances following instantaneous injections into a renal artery. Am. J. Physiol. 201, 795-798. Ch in ar d , F. P., an d En n s , T. (1955). Relative renal excretion patterns of sodium ion, .hloride ion, urea, water and glomerular substances. Am. J. Pkysiol. 182, 247-250. Cl ar k s o n , T. W., an d Ke n c h , J. E. (1956). Urinary excretion of amino acids by men ab sorbing heavy metals. Biochem. J. 62, 361-372. Co l l in s , R. J, Fr o mm, P. O., a n d Co l u n g s , W. D. (1961). Chromium excretion in the dog. Am. J. Physiol. 201, 795-798. '' Gay er , J., Gr au l , E. H., an d Hu n d es h ag en , H. (1962). Die Lokalisienmg des Transporter von Hg"-Ionen in der Niere durch Stop-flow-Analyse. Klin. Wockenschrifl 40, 953-955. Har r is o n , H. E. (1942). in "Principles of Renal Physiology" by H. W. Smith, p. 210. Oxford University Press, New York, 1957. Hu b s h , J. B, .Lo v aas , A, Pic c ib il l i, A. a n d Pu t n a m, T. E. (1960). Urinaiy excretion of radium in dogs. Am. J. Physiol. 199, 513-515. Ma l v in , E. L., Wil d e, W, S. a n d Su l u v a n , L. P. (1958). Localisation of nephron transport by stop-flow analysis. Am. J. Physiol. 194,135-140. Mambo u r g , A. M., an d Ray n au d , C. (1965). Etude a l'aide d'isotopes radioactifs, du mecaniisme de l'excretion urinaire du mercure chez le lapin. Rev. fran. etudes din. et biol. 10, 414-418. Pas s o w , H., Ro t h s t ein , A. a n d Cl a r k s o n , T. W. (1961). The general pharmacology of the heavy metals. Pharmacol. Rev. 13, 185-224. Ro d in , A. E., an d Cr o w s o n , C. N. (1962). Mercury nephrotoxicity in the rat. 1. Factors in fluencing the localisation of the tubular lesions. Am. J. Pathol. 61,297-313. Sal t man , P. (1965). The role of chelation in iron metabolism. J. Chem. Educ. 42, 682-687. Simmo n d s , J. P. (1942). Renal pathological changes in hypertension and glomerulonephritis. Clinical interpretation. J.'Am. Med. Assoc. 120, 89-93. Smit h , H. W. (1955). In "The kidney. Structure and function in health and disease," pp. 39-62. Oxford University Press, Nbw York. Sr k r ber , I. (1948). Investigations on the circulatory system of the avian kidney. Zeal. Bidray, Uppsala 27, 429-448. '----Th o el bu , H., a n d Pl et c h er , A. (1953). Behaviour of amino acids and enzymes in heavy metal poisoning. IH. Acute experimental mercuric poisoning. Helv. Physiol, et Pharmacol. Acta 11, 64-66. Van d er , A. J. (1962) Effect of cadmium on renal tubular sodium transport. Am. J. Physiol. 203, 1-5. Vo s t Al , J. (1963). Mechanisms of renal lead excretion. Biochem. Pharmacol. Conf. Issue 2. 207. Vo s t Al , J., an d Hel l er , J. (1963). Transtubular transport of solutes in the kidney. Pro ceedings, 2nd Int. Congress of Nephrology, Excerpta Medica Internal. Congress Ser. No. 7S. Amsterdam, pp. 130-132. Wans, J. H. (1953). Characteristics of uranium poisoning. In "The Pharmacology and Toxi cology of Uranium Compounds" (C. Voegtlin and H. C. Hodge, eds.) Vol. 1., pp. 237-2S0. McGraw-Hill, New York. mii i.n ml n t a i. id : Quantita II'iUi .l.v.-wiV. A rational* rmupurisot! oi .f ibe tcrlmii by ui.i jnlni-imli l-nmihtry bi-t (h i ihtermine Win n the -tnv-, eompul i-lutliimt nssei in work or ill -bips between t;i| or ililTiisiu The various util each otllCl tn ensure that u!i an extrein obtained at i :?.n' a burden c in importance mperuture. Si >p or jute suit ' et tiis skin fre A soldiers cl b'-'ing: protect ffiu builets at uni several oth 11 U to the eni ' rimm imjiosi * Active. i he purpose i'.-tuilf of 1 . -.I:-ifu-fj a ' Uehniqu DUP050312361